The formation of 3-oxo-delta4-steroids from 3xcex2-hydroxy-delta5 precursors in the biosynthesis of all classes of steroid hormones in mammals is catalysed by the enzyme system 3xcex2-hydroxy-delta5-steroid dehydrogenase (EC 1.1.1.145) and delta5-delta4-steroid isomerase (EC 5.3.3.1), designated 3xcex2-HSD. For example, 3xcex2-HSD catalyses the transformation of pregnenolone to progesterone, of 17xcex1-hydroxypregnenolone to 17xcex1-hydroxyprogesterone, of dehydroepiandrosterone to delta4-androstenedione or of 5-androstene-3xcex2-17xcex2-diol to testosterone (Simard et al., 1996).
Thus, 3xcex2-HSD is one of the key enzymes in the route for biosynthesis of hydrocortisone starting from cholesterol in the adrenal cortex of mammals (FIG. 1).
The use of recombinant microorganisms, especially modified yeasts, permitting heterologous expression of one or more of the mammalian enzymes of this biosynthetic route for producing hydrocortisone or intermediates of this biosynthesis was described for example in European patent application EP 340878, U.S. Pat. No. 5,137,822, Dumas et al., 1994 and Cauet et al., 1994.
When functional 3xcex2-HSD is expressed in yeast, the transformed yeast cells do not completely convert 3xcex2-hydroxysteroids to the corresponding 3-oxosteroids, for example pregnenolone to progesterone, but accumulate a compound which is also observed in the case of cells of untransformed yeast. Identification of the compound accumulated as the 3xcex2-acetate ester of the starting steroid and characterization of the enzyme possessing acyltransferase activity which is responsible for this esterification (designated hereinafter as APAT for xe2x80x9cacetyl-coenzyme A pregnenolone acetyltransferasexe2x80x9d) are described in the present application. Furthermore, accumulation of pregnenolone acetate by a pregnenolone-producing transformed yeast strain was described in European patent application EP 727489. It can be considered on the basis of these observations that esterification of the 3xcex2-hydroxysteroids produced by the yeast is undesirable as it is responsible for secondary reactions and by-products leading to a decrease in the yield of accumulated 3xcex2-hydroxysteroids, for example pregnenolone, or to a decrease in the yield of bioconversion of 3xcex2-hydroxy-delta5-steroids to 3-oxo-delta4-steroids, particularly in the production of progesterone or of 17xcex1-hydroxy-progesterone leading to a decrease in subsequent production of hydrocortisone by the biosynthetic route already mentioned.
On the basis of the results obtained, mentioned above, the present invention describes the construction of yeast strains that have lost the undesirable APAT activity, by alteration of the gene coding for this activity, resulting in stabilization of the 3xcex2-hydroxysteroids in the presence of the latter. These strains can therefore be used as starting strains for constructing recombinant strains that are capable of converting 3xcex2-hydroxysteroids to further products with improved yields.
The invention also describes the construction of yeast strains that have lost APAT activity by alteration of the gene coding for this activity and either expressing 3xcex2-HSD or the cytochrome P45017xcex1, or co-expressing 3xcex2-HSD and the cytochrome P45017xcex1 of the route of biosynthesis of hydrocortisone from cholesterol. The strains expressing for example 3xcex2-HSD make it possible to improve the yields in bioconversion of 3xcex2-hydroxy-delta5-steroids to 3-oxo-delta4-steroids and can therefore be used in processes of improved production of hydrocortisone or of its intermediates in yeast.
A subject of the present invention is therefore a modified yeast strain in which the acetyl-CoA pregnenolone acetyltransferase (APAT) activity is eliminated by altering the gene coding for this activity, resulting in stabilization of the 3xcex2-hydroxysteroids.
Alteration of the gene coding for APAT activity can be effected for example by insertion, deletion or substitution of a DNA sequence in the functional elements of the gene, for example the promoter or the sequence coding for the protein possessing APAT activity. Integration of the DNA sequence altered in this way in a host strain of yeast can then be effected for example by the technique of homologous recombination and leads to the generation of chromosomal mutants of yeast corresponding to the modified strains of the invention in which the disappearance of APAT activity and the stabilization of 3xcex2-hydroxysteroids are demonstrated, for example by cell culture in the presence of pregnenolone and by measuring the pregnenolone content as a function of time, following the operating conditions described later in the experimental section.
The following may be mentioned in particular as host yeast strains used for the invention: strains of Saccharomyces such as S. cerevisiae, strains of Candida such as C. maltosa, strains of Kluyveromyces such as K. lactis or strains of Pichia such as P. pastoris. 
A particular subject of the invention is a yeast strain modified as above in which the gene altered is the ATF2 gene of S. cerevisiae or a homologue of the latter.
By gene ATF2 we mean the gene of S. cerevisiae identified in the yeast genome at locus ATF2 or YGR177c, of xe2x80x9cSaccharomyces Genome Databasexe2x80x9d (SGD); (Cherry et al. http://genome-www.stanford.edu/Saccharomyces/) of which the open reading frame (ORF) designated YGR177c is translated into an amino acid sequence in the Mips database, accessible under accession number S64491 (Hebling U., Hofmann B. and Delius H. (May 1996)) and whose sequence is shown in FIG. 4. This gene codes for a protein possessing APAT activity, as is shown later in the experimental section.
By gene that is a homologue of the ATF2 gene, we mean a gene that codes for a protein possessing APAT activity and possessing sequence identity of about 60% or more with the sequence of protein YGR177C.
A more particular subject of the invention is a modified yeast strain as above in which the altered gene is the ATF2 gene of S. cerevisiae, designated hereinafter as atf2 mutant strain.
A quite particular subject of the invention is a modified yeast strain as above, in which the ATF2 gene is altered by insertion of a DNA sequence that has at least one nucleotide.
The DNA sequence that is inserted in the ATF2 gene so as to lose all APAT activity can be, for example, an auxotrophic selection gene supplying a nutritional requirement of the host strain such as the gene URA3, the gene LEU2, the gene TRP1, the gene HIS3 or the gene ADE2, for example a dominant selection gene such as a gene for resistance to an antibiotic such as G418, phleomycin or hygromycin B or for example a reporter gene such as the xcex2GAL gene.
The DNA sequence that is inserted in the ATF2 gene can also be a yeast expression block made up of a promoter and a transcription terminator, for example a yeast promoter such as PGK, TDH3, CYC1 or TEF1, for example a yeast terminator such as CYC1, TDH3, TEF1 or PGK. The expression block can be a combination of the elements mentioned above, for example the block TEF1prom/PGKterm.
A more quite particular subject of the invention is a modified yeast strain as above, in which the ATF2 gene is altered by insertion of the URA3 selection gene or of the expression block TEF1prom/GKterm.
A particular subject of the invention is a modified yeast strain as above, in which the ATF2 gene is altered by insertion of the URA3 selection gene.
The atf2 mutant strains of the invention, devoid of APAT activity and in which the URA3 gene has been inserted, designated hereinafter as atf2-xcex94::URA3, could thus be selected by prototrophy with uracil.
A quite particular subject of the invention is modified strains of S. cerevisiae designated as TGY156 and TGY158, the detailed constructions of which are given later in the experimental section.
A particular subject of the invention is also a modified yeast strain as above, in which the ATF2 gene is altered by insertion of the expression block TEF1prom/PGKterm, The atf2 mutant strains of the invention, devoid of APAT activity and in which the expression block TEF1prom/PGKterm has been inserted, designated hereinafter as atf2-xcex94::TEF1prom/PGKterm, could be selected for absence of a functional URA3 gene, replaced by an expression block, by their resistance to 5-fluoro-orotic acid (5-FO).
A quite particular subject of the invention is the modified strain of S. cerevisiae designated as TGY186, the detailed construction of which is given later in the experimental section.
A subject of the invention is also a transformed yeast strain in which the acetyl-CoA pregnenolone acetyltransferase (APAT) activity is eliminated by altering the gene coding for this activity and expressing at least one of the mammalian enzymes of the route of biosynthesis of hydrocortisone starting from cholesterol, chosen from:
the cholesterol side chain cleavage enzyme (P450SCC),
3xcex2-hydroxy-delta5-steroid dehydrogenase/delta5-delta4-steroid isomerase (3xcex2-HSD) and
17xcex1-steroid hydroxylase (P45017xcex1).
The transformed yeast strains of the invention can be obtained for example by transformation of atf2 mutant strains of the invention by known methods, for example by transformation by an expression vector of P450SCC as well as of ADX and ADR, by an expression vector of 3xcex2-HSD or by an expression vector of P45017xcex1. The atf2 mutant strains can also be co-transformed if necessary, for example by an expression vector of 3xcex2-HSD and by an expression vector of P45017xcex1 or be transformed by a co-expression vector of 3xcex2-HSD and P445017xcex1 and be used for example in a process of bioconversion of pregnenolone to 17xcex1-hydroxyprogesterone.
Vectors constructed for the expression of P450SCC as well as of ADX and ADR, of 3xcex2-HSD or of P45017xcex1 of bovine or human origin in yeast strains were described for example by Dumas et al., 1994, in European patent application EP 340878 or in U.S. Pat. No. 5,137,822.
A particular subject of the invention is a transformed yeast strain as above, in which the altered gene is the ATF2 gene of S. cerevisiae or a homologue of the latter. A more particular subject of the invention is a transformed yeast strain as above, in which the altered geneis, the ATF2 gene of S. cerevisiae and corresponds to a transformed atf2 strain.
A quite particular subject of the invention is a transformed yeast strain as above, in which the ATF2 gene is altered by insertion of a DNA sequence possessing at least one nucleotide and especially a transformed yeast strain in which the ATF2 gene is altered by insertion of the URA3 selection gene and corresponds to a modified atf2-xcex94::URA3 strain.
Alteration of the gene so as to lose all APAT activity, the ATF2 gene or a homologue of the latter as well as the host strains have the meanings indicated previously.
A quite particular subject of the invention is a transformed yeast strain atf2-xcex94::URA3 as above expressing 3xcex2-HSD and in particular the transformed strain of S. cerevisiae designated TGY158/pTG10862 of which a detailed construction is described later in the experimental section.
A quite particular subject of the is also a transformed yeast strain as above, in which the ATF2 gene is altered by insertion of the expression block TEF1prom/PGKterm and corresponding to a transformed strain atf2-xcex94::TEF1prom/PGKterm.
A quite particular subject of the invention is also a transformed strain atf2-xcex94::TEF1prom/PGKterm as above expressing P45017xcex1 and in particular the transformed strain of S. cerevisiae designated TGY186/pTG10435.
A particular subject of the invention is a modified yeast strain atf2-xcex94::TEF1prom/PGKterm as above co-expressing 3xcex2-HSD and P45017xcex1 and quite particularly the transformed strain of S. cerevisiae designated as TGY186/pTG10417.
A subject of the invention is also a process of oxidation in vivo of a substrate chosen among an endogenous sterol, an exogenous sterol or an exogenous steroid in which transformed yeast strain as above is used, which is either cultivated alone when the strain produces the endogenous sterol, or is incubated with the sterol or the exogenous steroid and the oxidized compound obtained is isolated if required.
By endogenous sterol we mean a sterol that is accumulated in a yeast strain and which is a substrate of the side chain cleavage enzyme (P450SCC) when the yeast, after transformation for example by an expression vector of P450SCC, of ADX and of ADR, is cultivated in the absence of exogenous sterol. The endogenous sterols used for applying the process of the invention can be for example ergosta-5-en-3-ol, ergosta-5,24(28)-dien-3-ol or ergosta-5,22-dien-3-ol. European patent application EP 727489 describes the accumulation of these sterols in a yeast strain and the cleavage of their side chain in a culture of the strain after transformation by an expression vector of P450SCC, of ADX and of ADR. Such a yeast strain, in which APAT activity is also present, can be modified beforehand to obtain an atf2 mutant strain according to the invention, then be transformed by an expression vector of P450SCC, of ADX and of ADR to obtain an atf2 mutant strain transformed according to the invention.
By exogenous sterol we mean a sterol which is a substrate of the P450SCC cleavage enzyme by incubation with a yeast strain transformed by an expression vector of P450SCC, of ADX and of ADR, for example cholesterol or sitosterol. Such a strain can be, for example, an atf2 mutant strain transformed by an expression vector of P450SCC, of ADX and of ADR.
The 3xcex2-hydroxysteroid obtained by cleavage of the side chain of the endogenous or exogenous sterol used as substrate is completely in the free form, i.e. is not accompanied by the corresponding 3xcex2-acetate ester, in cultures of transformed atf2 strains expressing P450SCC, ADX and ADR.
By steroid we mean a steroid which is a substrate of the 3xcex2-HSD enzyme by incubation with a yeast strain transformed for example by an expression vector of 3xcex2-HSD, such as pregnenolone, 17xcex1-hydroxypregnenolone or dehydroepiandrosterone or a steroid which is a substrate of the P45017xcex1 enzyme by incubation with a yeast strain transformed for example by an expression vector of P45017xcex1, such as progesterone or pregnenolone. Such a strain can, for example, be an atf2mutant strain transformed by an expression vector of 3xcex2-HSD or by an expression vector of P45017xcex1 according to the invention.
A particular subject of the invention is the in-vivo oxidation process as above, in which the substrate is a 3xcex2-hydroxysteroid and in which a transformed yeast strain atf2xcex94::URA3 is used, expressing 3xcex2-HSD, and the 3-oxo-delta4-steroid obtained is isolated if necessary, and especially a process in which the 3xcex2-hydroxysteroid is chosen from pregnenolone or 17xcex1-hydroxypregnenolone.
The 3xcex2-hydroxysteroid used as substrate is stable when it is incubated with an atf2-xcex94::URA3 strain of the invention, transformed by an expression vector of 3xcex2-HSD. The invention thus provides an improved process for production of 3-oxo-delta4-steroid in a yeast since all of the 3xcex2-hydroxy substrate can be oxidized to 3-oxo-delta4-steroid, as is shown later in the experimental section.
A particular subject of the invention is also the in-vivo oxidation process as above, in which the substrate is a steroid and in which a transformed yeast strain atf2-xcex94::TEF1prom/PGKterm expressing P40517xcex1 is used, and the 17xcex1-hydroxyl steroid obtained is isolated if necessary, and especially a process in which the steroid substrate is pregnenolone or progesterone.
The pregnenolone used as substrate is stable when it is incubated with a strain atf2-xcex94::TEF1prom/PGKterm transformed by an expression vector of P45017xcex1. The invention thus also provides an improved method of production of 17xcex1-hydroxysteroids starting from 3xcex2-hydroxysteroids since all of the 3xcex2-hydroxy substrate can be 17xcex1-hydroxylated.
A particular subject of the invention is also the above in-vivo oxidation process, in which the substrate is a 3xcex2-hydroxysteroid and in which a transformed yeast strain atf2-xcex94::TEF1prom/PGKterm co-expressing 3xcex2-HSD and P450-17xcex1 is used, and the 17xcex1-hydroxyl 3-oxo-delta4-steroid obtained is isolated if necessary. A quite particular subject of the invention is the above process in which the steroid substrate is pregnenolone.
The transformed atf2 mutant yeast strains and the process of the invention suggest their advantageous use in improved production of hydrocortisone or of its intermediates in yeast.
Examples of construction of the strains of the invention and of application of the process of the invention are described later in the experimental section.
1. Strains and Media
The strains of S. cerevisiae used for carrying out the invention are the strain TGY73.4 (MATxcex1, URA3-xcex945, pral-1, prbl-1, prcl-1, cpsl-3, his) isogenic derivative Leu+ of cl3ABYS86 described by Achstetter et al., 1992 and the strain Fyl679 (MATa, URA3-52, trp1-xcex9463, leu2-xcex941, his3-xcex94200, fen1, GAL) described by Thierry et al., 1990. The strains are grown on YPD complete medium (Difco Laboratories) containing 2% of glucose at 28xc2x0 C. according to the conditions described by F. Sherman, 1991.
For the transformation of S. cerevisiae, the cells are made competent by the lithium acetate method (Ito et al., 1983). The yeasts are cultivated routinely on a synthetic minimum medium SD containing 2% of glucose (F. Sherman, 1991) with addition of the required nutrients at a concentration of 100 xcexcg/ml.
The E. coli strain BJS183 (D. Hanahan, 1983) was used for in-vivo recombination and the E. coli strain C600, hsdR (Hubacek et al., 1970) was used as the receiving strain for the classical reactions of ligation.
2. Manipulation of the DNA and Recombination in vivo in E. coli 
The general methods of molecular biology used are described by Sambrook et al., 1989. The method for recombination in vivo was described by E. Degryse, 1995 and E. Degryse, 1996.
3. Test of APAT Enzyme Activity
The APAT acetyltransferase activity was determined by measuring the incorporation of [3H]acetate in the pregnenolone from [3H]acetyl-CoA (New England Nuclear). The reaction medium (500 xcexcl) contains [3H]acetyl-CoA (20 xcexcM, 25 Ci/mol) and pregnenolone (Sigma) (30 xcexcM). The pregnenolone is added in solution in 2 xcexcl of tyloxapol (Sigma)/ethanol mixture (1:1) in a potassium phosphate buffer (20 mM) at pH 7.0. After incubation for 15 minutes at 30xc2x0 C., the reaction is stopped by adding 2 ml of dichloromethane.
The steroids are extracted with dichloromethane, then separated by reversed-phase high-performance liquid chromatography (hereinafter: RP-HPLC) in isocratic elution conditions with acetonitrile in an Ultrasphere ODS column (Beckman) at 45xc2x0 C. on an HP 1090 chromatograph (Hewlett-Packard) connected to a FLO-One 500 radiodetector (Packard) which permits measurement of the amount of pregnenolone [3H]acetate formed.
One APAT unit is defined as the quantity of enzyme that produces 1 nmol of pregnenolone acetate per minute at 30xc2x0 C., in the conditions described above.
4. Determination of the Concentration of Protein
The protein concentration was measured using the xe2x80x9cprotein assay kitxe2x80x9d (Bio-Rad) with bovine serum albumin as standard.